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Vectors, mutant viruses and methods for generating mutant virusesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Vectors, mutant viruses and methods for generating mutant viruses description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070110720, Vectors, mutant viruses and methods for generating mutant viruses. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to nucleic acid vectors for delivery of a nucleic acid cassette to an insertion site in a selected viral genome, to methods of generating mutant virus using said vectors and to the mutant viruses generated, and particularly, although not exclusively, to mutant herpes simplex viruses and nucleic acid vectors for use in generating mutant herpes simplex viruses. BACKGROUND TO THE INVENTION [0002] Existing procedure for generating herpes simplex virus (HSV) mutants requires generation of a unique plasmid by cloning an entire expression cassette consisting of a promoter, gene of interest and polyadenylation sequences into a plasmid separately constructed to contain the relevant flanking sequences and then co-transfecting BHK cells with the resultant plasmid and HSV-1 DNA. Homologous recombination drives the formation of recombinant HSV-1 expressing the gene of interest, which is identified by Southern blotting. The recombinant virus is plaque purified 3-4 times by Southern blotting. This process takes several months. [0003] This approach was taken by Liu et al.sup.1 in generating two distinct plasmids, the first consisting of HSV-1 strain 17+ Sau3A fragment derived sequences flanking an expression cassette consisting of a CytoMegalovirus (CMV) promoter, Green Fluorescent Protein (GFP) gene and bGH polyadenylation (polyA) signal and the second wherein the GFP gene is replaced with either a human or mouse Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) gene. [0004] Shuttle vectors have been used to generate recombinant adenoviral vectors, e.g. the pAdEasy.TM. system of vectors (Stratagene), for use in overexpressing recombinant proteins in mammalian cells. However, these vectors require the cloning of the gene of interest into a first shuttle vector which is then co-transformed into a specially constructed cell line to generate a recombinant adenoviral plasmid which is transfected into a separate specially constructed mammalian cell line in which the recombinant adenoviral plasmid is directly packaged into virus particles. [0005] The HSV genome comprises two covalently linked segments, designated long (L) and short (S). Each segment contains a unique sequence flanked by a pair of inverted terminal repeat sequences. The long repeat (RL or R.sub.L) and the short repeat (RS or R.sub.S) are distinct. [0006] The HSV ICP34.5 (also .gamma.34.5) gene, which has been extensively studied.sup.1,6,7,8, has been sequenced in HSV-1 strains F.sup.9 and syn17+.sup.3 and in HSV-2 strain HG52.sup.4. One copy of the ICP34.5 gene is located within each of the RL repeat regions. Mutants inactivating both copies of the ICP34.5 gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 1716.sup.2 (HSV1716) or the mutants R3616 or R4009 in strain F.sup.5, are known to lack neurovirulence, i.e. be avirulent, and have utility as both gene delivery vectors or in the treatment of tumours by oncolysis. HSV1716 has a 759 bp deletion in each copy of the ICP34.5 gene located within the BamHI s restriction fragment of each RL repeat. [0007] ICP34.5 null mutants such as HSV1716 are, in effect, first-generation oncolytic viruses. Most tumours exhibit individual characteristics and the ability of a broad spectrum first generation oncolytic virus to replicate in or provide an effective treatment for all tumour types is not guaranteed. [0008] The prior art provides technically challenging, procedurally slow and inefficient materials and methods for generating recombinant HSV. In particular the prior art does not provide methods of, and materials for, generating recombinant HSV which are easy to detect, may be designed to be specific null mutants and which may express a selected gene of interest. [0009] First generation oncolytic viruses such as HSV-1 strain 17 mutant 1716 show significant therapeutic potential in tumour and gene therapy. Overcoming the existing technical difficulties by enabling rapid generation and screening of second generation oncolytic viruses of this kind provides a significant improvement in the development of novel pharmaceutical compositions, vaccines and medicaments for the treatment of cancer and disease. [0010] HSV 1716 is described in EP 0571410 and WO 92/13943 and has been deposited on 28 Jan. 1992 at the European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratories, Public Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number V92012803 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (herein referred to as the `Budapest Treaty`). SUMMARY OF THE INVENTION [0011] The inventors have provided a generic plasmid vector designated RL1.dIRES-GFP. RL1.dIRES-GFP provides a platform for generating a plurality of `shuttle vectors` which can exploit the process of homologous recombination to transfer a nucleotide sequence of interest (downstream of a selected promoter) into the disabling RL1 locus of HSV-1, generating easily identifiable, oncolytic, ICP34.5 null HSV-1 mutants expressing the products of the nucleotide sequence of interest, e.g. an RNA transcript or a polypeptide, and GFP. RL1.dIRES-GFP thus provides for ease of generation and purification of ICP34.5 null HSV. [0012] RL1.dIRES-GFP is a useful vector for making second-generation oncolytic viruses having enhanced cytotoxic potential and which may express the product(s) of selected gene(s) to enhance the oncolytic and/or therapeutic effect of the administered virus. [0013] The RL1.dIRES-GFP plasmid incorporates a multi-cloning sequence (MCS), upstream of an internal ribosome entry site (IRES), the GFP gene and SV40 polyadenylation sequences flanked by HSV-1 RL1 sequences. Incorporation of the encephalomyocarditis virus IRES (EMCV IRES) permits translation of two open reading frames from a single transcribed mRNA. [0014] Following generation of a specific shuttle vector by cloning of the nucleotide sequence of interest (and the selected promoter) into RL1.dIRES-GFP, recombinant HSV-1 expressing the desired nucleic acid transcript or protein, can be generated and purified within 2 weeks. This compares with 2-3 months using prior art protocols. [0015] In the ICP34.5 null HSV generated using the RL1.dIRES-GFP plasmid provided by the inventors transcription of both the nucleotide sequence of interest and GFP as a single transcript is controlled by the same promoter upstream of the nucleotide sequence of interest, the transcribed IRES directing cap-independent translation of GFP. The generated ICP34.5 null HSV are non-neurovirulent. By modifying the RL1.dIRES-GFP plasmid to incorporate appropriate flanking sequences surrounding the cassette other gene-specific HSV null mutants expressing GFP can be generated. [0016] RL1.dIRES-GFP is promoterless, thus enabling a promoter of choice to be incorporated in the homologously recombined shuttle vector for controlling expression of the nucleotide sequence of interest from the inserted cassette. [0017] Plasmid RL1.dIRES-GFP or modified plasmid shuttle vectors thereof further comprising nucleotide sequence encoding a nucleic acid transcript or polypeptide of interest may be provided in isolated or purified form. [0018] By using the plasmid RL1.dIRES-GFP to generate a shuttle vector, designated RL1.dCMV-NTR-GFP, containing the E. coli nitroreductase gene downstream of a CMV IE promoter, both inserted at the MCS, the inventors have further provided a novel second generation oncolytic mutant HSV. The genome of this mutant HSV comprises the heterologous (i.e. non-HSV) E. coli nitroreductase protein coding sequence inserted at one or each ICP34.5 locus, disrupting the ICP34.5 protein coding sequence such that the ICP34.5 gene is non-functional and cannot express a functional ICP34.5 gene product. The generated HSV is capable of expressing the E. coli nitroreductase gene product under control of the inserted promoter. This virus thus has the oncolytic activity of HSV strain 1716 and can be used in gene directed enzyme-prodrug therapy and has shown significantly enhanced tumour cell killing in vitro and in vivo when used with the prodrug CB1954. The mutant virus is designated HSV1716/CMV-NTR/GFP. [0019] As the plasmid RL1.dIRES-GFP is designed for tandem expression of a sequence of interest and the marker gene encoding green fluorescent protein (GFP). The sequence of interest is cloned into RL1.dIRES-GFP along with its promoter (e.g. CMV) such that the promoter drives transcription of an mRNA for the sequence of interest along with the IRES-GFP. Translation results in expression of the GFP from the internal ribosomal entry site and the gene of interest and promoter must be cloned into RL1.dIRES-GFP in the correct orientation to achieve this. There are a number of instances where this tandem expression arrangement may be unsuitable and a variation of the cassette design is favourable. [0020] One example is the expression of siRNAs as short hairpin RNAs using RNA polIII promoters such as H1 or U6. These promoters are unable to drive the additional tandem expression of the IRES-GFP as the RNApolIII expression cassette is designed only to produce short transcripts. Additionally, sequences of interest derived from genomic DNA with strong mRNA shut-off signals in their 3' untranslated regions may not support IRES-GFP expression. [0021] Thus in some cases a cassette may be provided in which the sequence of interest and marker are expressed separately from independent promoters. Continue reading about Vectors, mutant viruses and methods for generating mutant viruses... 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